Subunit Structure and Function in Vacuolar H+-ATPase

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DESCRIPTION (provided by applicant): Vacuolar proton-translocating ATPases (V-ATPases) couple hydrolysis of cytosolic ATP to proton transport into organelles of all eukaryotic cells and across the plasma membrane of some cell types. Organelle acidification, the major constitutive function of V-ATPases, is essntial for many physiological processes, but is also linked to a number of disease states. For example, acidification of phagosomes is essential for killing invading bacteria, but many viruses and toxins exploit the acidic environment generated by V-ATPases to facilitate their escape from organelles into the cytoplasm where they become biologically active. Plasma membrane V-ATPases are involved in renal acid secretion and osteoclast bone dissolution; mutations in tissue-specific V-ATPase subunit isoforms necessary for these processes result in genetic diseases characterized by metabolic acidosis and osteoporosis. The long-term goal of the lab is to understand the structure, function, assembly and regulation of V-ATPases by studying the yeast V-ATPase, which has proven to be an excellent model for all eukaryotic V-ATPases. All V-ATPases are composed of two multisubunit domains, a peripheral membrane complex involved in ATP hydrolysis and an integral membrane complex required for proton transport. In this proposal, we focus on the "stalk" subunits that structurally and functionally bridge these two domains. These subunits are responsible for transmission of conformational changes resulting from ATP hydrolysis to the proton pore, and are also key players in regulated disassembly of V-ATPases, an important regulatory mechanism. The aims of this proposal are: 1) to position the stalk subunits in the yeast V-ATPase, by a combination of electron microscopy, hydrodynamic studies of subcomplexes, mutagenesis, and crosslinking experiments, 2) to elucidate the roles of the C and H subunits, particularly in the functionally important conformational change accompanying release of the peripheral sector from the membrane sector, 3) to examine protein-protein interactions with two isoforms of the "a" subunit and test their importance in regulated disassembly, and 4) to follow V-ATPase assembly and disassembly in vivo using GFP-tagged V-ATPase subunits.